The long-term goal of this research is to understand the molecular and mechanistic basis of G protein activation by an activated receptor. We will investigate the conformational changes that the G protein ? subunit undergoes as it interacts with activated rhodopsin, ultimately leading to GDP release, GTP binding, and activation. We will define these movements using a strategy of local sensors of conformational change using site-directed Cys mutagenesis to label particular sites with fluorophores and spin-labels. Additional information will be gained from labeling two sites and measuring the distances between them in different conformations using double electron-electron resonance (DEER). In the last funding cycle, we showed by DEER studies that there is a large opening of the cleft between GTPase and helical domains of G? triggered by the C-terminal helix contact with receptor, but the detailed mechanisms of the trigger of this large conformational change are unknown. In this grant period, we will seek (in specific aim 1) to elucidate how GDP release is coupled to conformational changes in regions involved in nucleotide binding. We will examine the dynamics of conformational changes on G? using an atomic resolution model that incorporates both the energetic as well as the DEER distance distributions. This model provides insight into how the bound GDP stabilizes the closed state as well as how the receptor triggers GDP release and domain opening. In specific aim 2, we will investigate the receptor-mediated conformational changes in the G? subunit which lead to domain opening. Finally, in specific aim 3, we will determine the mechanisms by which GTP binding leads to dissociation from R* and G protein activation. These studies will lead to a more complete picture of the membrane-bound signaling proteins in their native states, undergoing the conformational changes that lead to G protein activation. Signaling through this class of receptors underlies most aspects of our physiology and behavior, and many pathologies as well, and these studies may provide insights into how we might disrupt signaling through particular GPCR-G protein interactions in a number of disease states.